The experiments described in this proposal are designed to gain insights into the mechanism regulation of pre-RNA splicing in vertebrate cells. The work focuses on members of a family of conserved splicing factors known as SR proteins, but other proteins that participate in splicing and/or its regulation will also be analyzed. Studies examining the roles of snRNAs in splicing catalysis will be continued. The following specific aims are proposed. 1. Novel functions of ASF/SF2. The recent discovery that ASF/SF2 is necessary for the maintenance of genome stability by preventing mutagenic co-transcriptional R loops will be pursued in detail. In vitro assays to investigate the mechanism and specificity of RNP formation will be developed, with the ultimate goal of recreating R-loop formation and the subsequent DMA double strand breaks (DSBs) that occur in the absence of proper RNP formation and/or splicing. The mechanism of DMA DSB formation will also be investigated. A genetic screen to identify suppressors of ASF/SF2-induced cell death will be continued. 2. Properties of SRp38. The discovery that SRp38 can function as a sequence-specific activator of splicing will be pursued and the identity of an SRp38-specific co-activator determined. Analysis of the signaling pathway that leads to SRp38-dephosphorylation will be investigated. The mechanism by which Hsp27 functions to prevent SRp38 dephosphorylation and induce splicing thermo tolerance will be pursued. Analysis of a recently created SRp38 knockout mouse will be continued. 3. Other splicing regulators. The mechanism by which hnRNP A1 contributes to SMN2 exon 7 exclusion will be analyzed. The possibility that creation of hnRNP A1-dependent exonic splicing silencers by mutation, for example in BRCA1 and BRCA2 transcripts, occurs relatively frequently will be investigated. The identity of an RNAPII CTD-specific splicing co-activator will be established and its mechanism of action analyzed. The basis for the requirement of a PPP family phosphatase for the second catalytic step of splicing will be determined. 4. snRNAs and splicing catalysis. Characterization of RNA-Y, a novel product formed with purified segments of U2 and U6 RNAs in a reaction very similar to the first step of splicing, will be completed. Collaborative efforts to determine the crystal structure of the active U2/U6 complex will be continued. Experiments to identify protein cofactors that facilitate U2/U6- mediated catalysis and/or U2/U6 complex formation will be pursued. Select residues in U2/U6 will be subjected to mutagenesis to elucidate how they function in catalysis.